Testing the limits of multiplex respiratory virus assays for SARS-CoV-2 at high cycle threshold values: Comparative performance of cobas 6800/8800 SARS-CoV-2 & Influenza A/B, Xpert Xpress SARS-CoV-2/Flu/RSV, and cobas Liat SARS-CoV-2 & Influenza A/B.

Background: Multiplex real-time RT-PCR assays for respiratory pathogens are valuable tools to optimize laboratory workflow and turnaround time. At a time when resurgence of influenza and respiratory syncytial virus (RSV) cases have been widely observed along with continued transmission of SARS-CoV-2, timely identification of all circulating respiratory viruses is crucial. This study evaluates the detection of low viral loads of SARS-CoV-2 by four multiplex molecular assays: Roche cobas 6800/8800 SARS-CoV-2 & Influenza A/B Test, Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV, cobas Liat SARS-CoV-2 & Influenza A/B, and a laboratory-developed test (LDT). Methods: Retrospective upper respiratory tract specimens positive for various respiratory viruses at a range of cycle threshold (Ct) values (18-40) were tested by four multiplex assays. Positive and negative percent agreement (PPA and NPA) with validated RT-PCR assays were calculated. Results: A total of 82 samples were assessed, with discordant results observed in a portion of the samples (10/82, 12.2%) where Ct values were >33. The majority of the discordant results (6/10, 60%) were false negatives. Overall, PPA was 100% (58/58) for cobas 6800, 97.4% (38/39) for GeneXpert, 100% (17/17) for Liat, and 90.5% (57/63) for the LDT. PPA for the LDT increased to 92.1% after manual review of amplification curves. Conclusions: Commercial multiplex respiratory virus assays have good performance for samples with medium to high viral loads (Ct values <33). Laboratories should consider appropriate test result review and confirmation protocols to optimize sensitivity, and may consider reporting samples with additional interpretive comments when low viral loads are detected.

Historique: Les dosages multiplex par RT-PCR en temps réel (amplification en chaîne par polymérase avec transcription inverse en temps réel) des agents pathogènes respiratoires sont des outils précieux pour optimiser le flux de travail et le temps de traitement en laboratoire. Alors qu'on observe une résurgence générale des cas d'influenza et du virus respiratoire syncytial (VRS) et une transmission continue du SRAS-CoV-2, il est crucial de détecter rapidement tous les virus respiratoires en circulation. Dans la présente étude, les chercheurs ont évalué la détection des faibles charges virales du SRAS-CoV-2 à l'aide de quatre dosages moléculaires multiplex : le test cobas 6800/8800 SRAS-CoV-2 et influenza A/B de Roche, le test Xpress SRAS-CoV-2/influenza/VRS de Cepheid Xpert, le test cobas SRAS-CoV-2 et influenza A/B de Liat et un test créé par le laboratoire (TCL). Méthodologie: Les chercheurs ont procédé au dépistage rétrospectif d'échantillons ayant obtenu un résultat positif à divers virus respiratoires à une série de valeurs de cycle seuil (Ct) (18­40) à l'aide de quatre dosages multiplex. Ils ont calculé le pourcentage de concordance positif (PCP) et négatif (PCN) avec les dosages par RT-PCR validés. Résultats: Au total, les chercheurs ont évalué 82 échantillons et observé des résultats discordants dans une partie des échantillons (dix sur 82, 12,2 %), pour lesquels les valeurs Ct étaient supérieures à 33. La majorité de ces résultats discordants (six sur dix, 60 %) étaient faussement négatifs. Dans l'ensemble, le PCP atteignait 100 % (58 sur 58) selon le test cobas 6800, 97,4 % (38 sur 39) selon le test GeneXpert, 100 % (17 sur 17) selon le test Liat et 90,5 % (57 sur 63) selon le TCL. Le PCP du TCL est passé à 92,1 % après l'examen manuel des courbes d'amplification. Conclusions: Les dosages multiplex commerciaux des virus respiratoires donnent un bon rendement pour les échantillons contenant une charge virale modérée à élevée (valeurs Ct inférieures à 33). Les laboratoires devraient envisager de procéder à une analyse des résultats du dépistage et à des protocoles de confirmation appropriés pour en optimiser la sensibilité et pourraient également envisager d'ajouter des commentaires interprétatifs aux résultats des échantillons lorsque la charge virale décelée est faible.

Evaluating in vivo effectiveness of sotrovimab for the treatment of Omicron subvariant BA.2 versus BA.1: a multicentre, retrospective cohort study.

BACKGROUND: In vitro data suggested reduced neutralizing capacity of sotrovimab, a monoclonal antibody, against Omicron BA.2 subvariant. However, limited in vivo data exist regarding clinical effectiveness of sotrovimab for coronavirus disease 2019 (COVID-19) due to Omicron BA.2. METHODS: A multicentre, retrospective cohort study was conducted at three Canadian academic tertiary centres. Electronic medical records were reviewed for patients ≥ 18 years with mild COVID-19 (sequencing-confirmed Omicron BA.1 or BA.2) treated with sotrovimab between February 1 to April 1, 2022. Thirty-day co-primary outcomes included hospitalization due to moderate or severe COVID-19; all-cause intensive care unit (ICU) admission, and all-cause mortality. Risk differences (BA.2 minus BA.1 group) for co-primary outcomes were adjusted with propensity score matching (e.g., age, sex, vaccination, immunocompromised status). RESULTS: Eighty-five patients were included (15 BA.2, 70 BA.1) with similar baseline characteristics between groups. Adjusted risk differences were non-statistically significant between groups for 30-day hospitalization (- 14.3%; 95% confidence interval (CI): - 32.6 to 4.0%), ICU admission (- 7.1%; 95%CI: - 20.6 to 6.3%), and mortality (- 7.1%; 95%CI: - 20.6 to 6.3%). CONCLUSIONS: No differences were demonstrated in hospitalization, ICU admission, or mortality rates within 30 days between sotrovimab-treated patients with BA.1 versus BA.2 infection. More real-world data may be helpful to properly assess sotrovimab's effectiveness against infections due to specific emerging COVID-19 variants.

Stability of BK polyomavirus DNA in urine over time and analytical evaluation of an automated BKV quantitative nucleic acid test.

Introduction. BK polyomavirus (BKPyV) quantitative testing is an important screening tool post-transplantation, although interpretation can be challenging due to lack of standardization, assay heterogeneity and variability of BKPyV DNA over time (in urine).Methods. Remnant clinical EDTA plasma and urine samples were tested by the cobas BKV test and a validated laboratory-developed test (LDT). Accuracy [positive and negative percent agreement (PPA and NPA), Pearson's correlation, Bland-Altman analysis] and reproducibility were evaluated. To assess BKPyV DNA stability in urine, prospective urine samples were maintained at two different storage temperatures and tested in triplicate over 7 days.Results. Overall PPA was 95.6 % (43/45) and NPA was 94.4 % (170/180). For plasma, Pearson's correlation (0.950) and Bland-Altman analysis (0.113±0.22 log10 IU ml-1) showed high agreement. For neat urine, Pearson's correlation (0.842) and Bland-Altman analysis (0.326±0.80 log10 IU ml-1) showed somewhat higher variability. Reproducibility was high for the cobas BKV versus the LDT. BKPyV DNA levels in neat urine remained relatively stable over 7 days at both storage temperatures, although outlier results were intermittently detected.Conclusion. The cobas BKV test showed high agreement and reproducibility compared to the reference LDT. BKPyV viral load testing in urine has known limitations, but neat urine can be processed by the cobas BKV.

Effects of COVID-19 mRNA vaccination on HIV viremia and reservoir size.

OBJECTIVE: The immunogenic nature of COVID-19 mRNA vaccines led to some initial concern that these could stimulate the HIV reservoir. We analyzed changes in plasma HIV loads (pVL) and reservoir size following COVID-19 mRNA vaccination in 62 people with HIV (PWH) receiving antiretroviral therapy (ART), and analyzed province-wide trends in pVL before and after the mass vaccination campaign. DESIGN: Longitudinal observational cohort and province-wide analysis. METHODS: 62 participants were sampled pre-vaccination, and one month after their first and second COVID-19 immunizations. Vaccine-induced anti-SARS-CoV-2-Spike antibodies in serum were measured using the Roche Elecsys Anti-S assay. HIV reservoirs were quantified using the Intact Proviral DNA Assay; pVL were measured using the cobas 6800 (LLOQ:20 copies/mL). The province-wide analysis included all 290,401 pVL performed in British Columbia, Canada between 2012-2022. RESULTS: Pre-vaccination, the median intact reservoir size was 77 (IQR:20-204) HIV copies/million CD4+ T-cells, compared to 74 (IQR:27-212) and 65 (IQR:22-174) post-first and -second dose, respectively (all comparisons p>0.07). Pre-vaccination, 82% of participants had pVL<20 copies/mL (max:110 copies/mL), compared to 79% post-first dose (max:183 copies/mL) and 85% post-second dose (max:79 copies/mL) (p > 0.4). There was no evidence that the magnitude of the vaccine-elicited anti-SARS-CoV-2-Spike immune response influenced pVL nor changes in reservoir size (p > 0.6). We found no evidence linking the COVID-19 mass vaccination campaign to population-level increases in detectable pVL frequency among all PWH in the province, nor among those who maintained pVL suppression on ART. CONCLUSION: We found no evidence that COVID-19 mRNA vaccines induced changes in HIV reservoir size nor plasma viremia.

WGS of a cluster of MDR Shigella sonnei utilizing Oxford Nanopore R10.4.1 long-read sequencing.

OBJECTIVES: To utilize long-read nanopore sequencing (R10.4.1 flowcells) for WGS of a cluster of MDR Shigella sonnei, specifically characterizing genetic predictors of antimicrobial resistance (AMR). METHODS: WGS was performed on S. sonnei isolates identified from stool and blood between September 2021 and October 2022. Bacterial DNA from clinical isolates was extracted on the MagNA Pure 24 and sequenced on the GridION utilizing R10.4.1 flowcells. Phenotypic antimicrobial susceptibility testing was interpreted based on CLSI breakpoints. Sequencing data were processed with BugSeq, and AMR was assessed with BugSplit and ResFinder. RESULTS: Fifty-six isolates were sequenced, including 53 related to the cluster of cases. All cluster isolates were identified as S. sonnei by sequencing, with global genotype 3.6.1.1.2 (CipR.MSM5), MLST 152 and PopPUNK cluster 3. Core genome MLST (cgMLST, examining 2513 loci) and reference-based MLST (refMLST, examining 4091 loci) both confirmed the clonality of the isolates. Cluster isolates were resistant to ampicillin (blaTEM-1), trimethoprim/sulfamethoxazole (dfA1, dfrA17; sul1, sul2), azithromycin (ermB, mphA) and ciprofloxacin (gyrA S83L, gyrA D87G, parC S80I). No genomic predictors of resistance to carbapenems were identified. CONCLUSIONS: WGS with R10.4.1 enabled rapid sequencing and identification of an MDR S. sonnei community cluster. Genetic predictors of AMR were concordant with phenotypic antimicrobial susceptibility testing.

Hepatitis B virus genotype surveillance in Canadian blood donors and a referred patient population, 2016-2021.

BACKGROUND AND OBJECTIVES: Hepatitis B virus (HBV) genotypes (A-H) have a distinct geographic distribution and are highly associated with the country of birth. Canada has experienced increased immigration over the past decade, primarily from regions where HBV is endemic. This study investigated the proportions and trends of HBV genotypes within blood donor and clinical populations of Canada over the period 2016-2021. MATERIALS AND METHODS: Study samples involved two cohorts: (1) Canadian blood donors (n = 246) deferred from donation due to HBV test positivity and (2) chronic HBV patients from across Canada (clinically referred population, n = 3539). Plasma or serum was extracted, and the surface antigen and/or polymerase-coding region was amplified and sequenced to determine genotype by phylogenetic analysis. RESULTS: Six (A-E, G) and eight (A-H) HBV genotypes were detected among deferred blood donors and the clinically referred population, respectively. Differences in HBV genotype proportions between the two cohorts were observed across Canada. Males comprised most of the referred population among genotypes A-E (p < 0.0001), except for genotypes B and C. The median age was younger among blood donors (36 years [range 17-72]) compared with the referred population (41 years [range 0-99]). Distinct trends of increasing (E, referred; B, blood donor) and decreasing genotype prevalence were observed over the study period. CONCLUSION: HBV genotypes in Canada are highly diverse, suggesting a large immigrant population. Observed trends in genotype prevalence and proportional differences among cohorts imply shifts among the HBV-infected population of Canada, which warrants continued surveillance.

Detection of multiple human enteropathogens in Norway rats (Rattus norvegicus) from an under-resourced neighborhood of Vancouver, British Columbia.

Urban Norway rats (Rattus norvegicus) can carry various human pathogens, and may be involved in pathogen propagation and transmission to humans. From January 31-August 14, 2021, a community outbreak of Shigella flexneri serotype 2a occurred among unhoused or poorly housed people in the Downtown Eastside neighborhood of Vancouver, British Columbia, Canada. The source could not be identified; however, patients reported contact with rats, and previous studies indicated transmission of rat-associated zoonotic pathogens among the unhoused or poorly housed residents of this neighborhood. The study objective was to determine if rats trapped in the outbreak area were carriers of Shigella spp. and other zoonotic enteric pathogens. From March 23-April 9, 2021, 22 rats were lethally trapped within the outbreak area. Colonic content was analyzed using the BioFire FilmArray Gastrointestinal (multiplex PCR) panel for human enteropathogens, which detected: Campylobacter spp. (9/22), Clostridioides difficile (3/22), Yersinia enterocolitica (5/22), Cryptosporidium spp. (8/22), Giardia duodenalis (5/22), Rotavirus A (1/22), enteroaggressive Escherichia coli (2/22), enteropathogenic E. coli (10/22), and Shigella spp. or enteroinvasive E. coli (EIEC) (3/22). An ipaH PCR assay was used for targeted detection of Shigella spp./EIEC, with five rats positive. Two samples contained insertion sites unique to S. flexneri isolated from the human outbreak. This study highlights the potential for rats to carry a broad range of human pathogens, and their possible role in pathogen maintenance and/or transmission.

Effects of COVID-19 mRNA vaccination on HIV viremia and reservoir size.

Objective: The immunogenic nature of COVID-19 mRNA vaccines led to some initial concern that these could stimulate the HIV reservoir. We analyzed changes in plasma HIV loads (pVL) and reservoir size following COVID-19 mRNA vaccination in 62 people with HIV (PWH) receiving antiretroviral therapy (ART), and analyzed province-wide trends in pVL before and after the mass vaccination campaign. Design: Longitudinal observational cohort and province-wide analysis. Methods: 62 participants were sampled pre-vaccination, and one month after their first and second COVID-19 immunizations. Vaccine-induced anti-SARS-CoV-2-Spike antibodies in serum were measured using the Roche Elecsys Anti-S assay. HIV reservoirs were quantified using the Intact Proviral DNA Assay; pVL were measured using the cobas 6800 (LLOQ:20 copies/mL). The province-wide analysis included all 290,401 pVL performed in British Columbia, Canada between 2012-2022. Results: Pre-vaccination, the median intact reservoir size was 77 (IQR:20-204) HIV copies/million CD4+ T-cells, compared to 74 (IQR:27-212) and 65 (IQR:22-174) post-first and -second dose, respectively (all comparisons p>0.07). Pre-vaccination, 82% of participants had pVL<20 copies/mL (max:110 copies/mL), compared to 79% post-first dose (max:183 copies/mL) and 85% post-second dose (max:79 copies/mL) (p>0.4). The magnitude of the vaccine-elicited anti-SARS-CoV-2-Spike antibody response did not correlate with changes in reservoir size nor detectable pVL frequency (p>0.6). We found no evidence linking the COVID-19 mass vaccination campaign to population-level increases in detectable pVL frequency among all PWH in the province, nor among those who maintained pVL suppression on ART. Conclusion: We found no evidence that COVID-19 mRNA vaccines induced changes in HIV reservoir size nor plasma viremia.

Epidemiology of cytomegalovirus antiviral resistance testing for solid organ and bone marrow transplant patients from 2011 - 2019.

BACKGROUND: CMV reactivation post-transplantation is common, with need for prompt identification of patients most at-risk for CMV antiviral drug resistance (AVDR). OBJECTIVES: This study describes CMV AVDR frequencies, antiviral prescribing practices, and AVDR risk factors in patients from 2011 to 2019 in British Columbia, Canada. STUDY DESIGN: Retrospective review of demographics, transplant type, viral loads, antiviral exposure duration, and 12-month mortality was conducted for all patients with samples submitted for CMV AVDR testing from 2011 to 2019. Genotyping of AVDR mutations occurred at the national reference laboratory. Mann-Whitney U, T-test or Fisher's exact tests examined differences between patients with and without AVDR. RESULTS: Fifty-three plasma and three tissue/fluid specimens successfully underwent CMV AVDR testing; of these samples, 27/56 (48%) had AVDR mutations detected. The commonest AVDR mutations were at UL97 loci A594 (20%), H596 (12%) and L595 (12%). Mutations occurred more frequently in requests from solid organ than hematopoietic stem cell transplant patients (58% vs. 27%, p = 0.05). Previous resistance testing was a significant risk factor for AVDR (p < 0.001). Patients with AVDR had approximately 51 more days of antiviral therapy (p = 0.007) and took 9 days longer to clear viremia (p = 0.23). The median turnaround time from sample send-out to reporting was nine days. However, empiric use of second-line antivirals occurred in most cases (39/53, 74%) before results were available. DISCUSSION: Laboratories should strive to provide timely CMV AVDR testing for transplant patients, to minimize unnecessary exposure to second-line antiviral agents. The findings of this study may help guide clinicians when selecting empiric antiviral therapy.

No Isolate, No Problem: Using a Novel Insertion Sequence PCR to Link Rats to Human Shigellosis Cases in an Underserved Urban Community.

During an investigation into a cluster of Shigella flexneri serotype 2a cases in an underserved community, we assessed the relatedness of human and rat S. flexneri isolates utilizing a novel PCR targeting insertion sites (IS-PCR) of mobile elements in the Shigella genome characteristic of the cluster strain. Whole-genome sequences of S. flexneri (n = 50) associated with the cluster were analyzed. De novo genome assemblies were analyzed by a Geneious V10.2.6 motif search, and two unique IS were identified in all human Shigella sequences of the local cluster. Hydrolysis probe PCR assays were designed to detect these sequences consisting of forward and reverse primers to amplify across each insertion site and a hydrolysis probe spanning the insertion site. IS-PCR was performed for three Shigella PCR-positive culture-negative rat intestine specimens from this community. Both insertion sites were detected in the de novo genome assemblies of all clinical S. flexneri isolates (n = 50). Two of the three PCR-positive culture-negative rat samples were positive for both unique ISs identified in the human S. flexneri isolates, suggesting that the rat Shigella species strains were closely related to the human strains in the cluster. The cycle threshold (Ct) values were >35, indicating that the bacterial load was very low in the rat samples. Two unique IS were identified in clinical isolates from a community S. flexneri cluster. Both IS targets were identified in PCR-positive (Shigella spp.), culture-negative rat tissue and clinical isolates from humans, indicating relatedness. IMPORTANCE This article describes a novel molecular method to show relatedness between bacterial infections, which may not be able to grow in the laboratory due to treatment with antibiotics or for bacteria requiring unique conditions to grow well. Uniquely, we applied this technique to Shigella isolates from human cases associated with a local cluster in an underserved community, as well as rat samples from the same community. We believe that this novel approach can serve as a complementary method to support outbreak/cluster investigation for Shigella spp.

Impact of Age and Severe Acute Respiratory Syndrome Coronavirus 2 Breakthrough Infection on Humoral Immune Responses After Three Doses of Coronavirus Disease 2019 mRNA Vaccine.

Background: Longer-term immune response data after 3 doses of coronavirus disease 2019 (COVID-19) mRNA vaccine remain limited, particularly among older adults and after Omicron breakthrough infection. Methods: We quantified wild-type- and Omicron-specific serum immunoglobulin (Ig)G levels, angiotensin-converting enzyme 2 displacement activities, and live virus neutralization up to 6 months after third dose in 116 adults aged 24-98 years who remained COVID-19 naive or experienced their first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during this time. Results: Among the 78 participants who remained COVID-19 naive throughout follow up, wild-type- and Omicron-BA.1-specific IgG concentrations were comparable between younger and older adults, although BA.1-specific responses were consistently significantly lower than wild-type-specific responses in both groups. Wild-type- and BA.1-specific IgG concentrations declined at similar rates in COVID-19-naive younger and older adults, with median half-lives ranging from 69 to 78 days. Antiviral antibody functions declined substantially over time in COVID-19-naive individuals, particularly in older adults: by 6 months, BA.1-specific neutralization was undetectable in 96% of older adults, versus 56% of younger adults. Severe acute respiratory syndrome coronavirus 2 infection, experienced by 38 participants, boosted IgG levels and neutralization above those induced by vaccination alone. Nevertheless, BA.1-specific neutralization remained significantly lower than wild-type, with BA.5-specific neutralization lower still. Higher Omicron BA.1-specific neutralization 1 month after third dose was an independent correlate of lower SARS-CoV-2 infection risk. Conclusions: Results underscore the immune benefits of the third COVID-19 mRNA vaccine dose in adults of all ages and identify vaccine-induced Omicron-specific neutralization as a correlate of protective immunity. Systemic antibody responses and functions however, particularly Omicron-specific neutralization, decline rapidly in COVID-19-naive individuals, particularly in older adults, supporting the need for additional booster doses.

SARS-CoV-2 live virus neutralization after four COVID-19 vaccine doses in people with HIV receiving suppressive antiretroviral therapy.

OBJECTIVE: Limited data exist regarding the immune benefits of fourth COVID-19 vaccine doses in people with HIV (PWH) receiving antiretroviral therapy (ART), particularly now that most have experienced a SARS-CoV-2 infection. We quantified wild-type, Omicron-BA.5 and Omicron-BQ.1-specific neutralization up to 1 month post-fourth COVID-19 vaccine dose in 63 (19 SARS-CoV-2-naive and 44 SARS-CoV-2-experienced) PWH. DESIGN: A longitudinal observational cohort. METHODS: Quantification of wild-type-, Omicron-BA.5, and Omicron-BQ.1-specific neutralization using live virus assays. RESULTS: Participants received monovalent (44%) and bivalent (56%) mRNA fourth doses. In COVID-19-naive PWH, fourth doses enhanced wild-type and Omicron-BA.5-specific neutralization modestly above three-dose levels ( P  = 0.1). In COVID-19-experienced PWH, fourth doses enhanced wild-type specific neutralization modestly ( P  = 0.1) and BA.5-specific neutralization substantially ( P  = 0.002). Consistent with humoral benefits of 'hybrid' immunity, COVID-19-experienced PWH exhibited the highest neutralization post-fourth dose, wherein those with Omicron-era infections displayed higher wild-type specific ( P  = 0.04) but similar BA.5 and BQ.1-specific neutralization than those with pre-Omicron-era infections. Nevertheless, BA.5-specific neutralization was significantly below wild-type in everyone regardless of COVID-19 experience, with BQ.1-specific neutralization lower still (both P  < 0.0001). In multivariable analyses, fourth dose valency did not affect neutralization magnitude. Rather, an mRNA-1273 fourth dose (versus a BNT162b2 one) was the strongest correlate of wild-type specific neutralization, while prior COVID-19, regardless of pandemic era, was the strongest correlate of BA.5 and BQ.1-specific neutralization post-fourth dose. CONCLUSION: Fourth COVID-19 vaccine doses, irrespective of valency, benefit PWH regardless of prior SARS-CoV-2 infection. Results support recommendations that all adults receive a fourth COVID-19 vaccine dose within 6 months of their third dose (or their most recent SARS-CoV-2 infection).

Principles of diagnostic stewardship: A practical guide from the Society for Healthcare Epidemiology of America Diagnostic Stewardship Task Force.

We provide an overview of diagnostic stewardship with key concepts that include the diagnostic pathway and the multiple points where interventions can be implemented, strategies for interventions, the importance of multidisciplinary collaboration, and key microbiologic diagnostic tests that should be considered for diagnostic stewardship. The document focuses on microbiologic laboratory testing for adult and pediatric patients and is intended for a target audience of healthcare workers involved in diagnostic stewardship interventions and all workers affected by any step of the diagnostic pathway (ie, ordering, collecting, processing, reporting, and interpreting results of a diagnostic test). This document was developed by the Society for Healthcare Epidemiology of America Diagnostic Stewardship Taskforce.

End-to-End Data Automation for Pooled Sample SARS-CoV-2 Using R and Other Open-Source Tools.

BACKGROUND: Due to supply chain shortages of reagents for real-time (RT)-PCR for SARS-CoV-2 and increasing demand on technical staff, an end-to-end data automation strategy for SARS-CoV-2 sample pooling and singleton analysis became necessary in the summer of 2020. METHODS: Using entirely open source software tools-Linux, bash, R, RShiny, ShinyProxy, and Docker-we developed a modular software application stack to manage the preanalytical, analytical, and postanalytical processes for singleton and pooled testing in a 5-week time frame. RESULTS: Pooling was operationalized for 81 days, during which time 64 pooled runs were performed for a total of 5320 sample pools and approximately 21 280 patient samples in 4:1 format. A total of 17 580 negative pooled results were released in bulk. After pooling was discontinued, the application stack was used for singleton analysis and modified to release all viral RT-PCR results from our laboratory. To date, 236 109 samples have been processed avoiding over 610 000 transcriptions. CONCLUSIONS: We present an end-to-end data automation strategy connecting 11 devices, one network attached storage, 2 Linux servers, and the laboratory information system.

People With Human Immunodeficiency Virus Receiving Suppressive Antiretroviral Therapy Show Typical Antibody Durability After Dual Coronavirus Disease 2019 Vaccination and Strong Third Dose Responses.

BACKGROUND: Longer-term humoral responses to 2-dose coronavirus disease 2019 (COVID-19) vaccines remain incompletely characterized in people living with human immunodeficiency virus (HIV) (PLWH), as do initial responses to a third dose. METHODS: We measured antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, angiotensin-converting enzyme 2 (ACE2) displacement, and viral neutralization against wild-type and Omicron strains up to 6 months after 2-dose vaccination, and 1 month after the third dose, in 99 PLWH receiving suppressive antiretroviral therapy and 152 controls. RESULTS: Although humoral responses naturally decline after 2-dose vaccination, we found no evidence of lower antibody concentrations or faster rates of antibody decline in PLWH compared with controls after accounting for sociodemographic, health, and vaccine-related factors. We also found no evidence of poorer viral neutralization in PLWH after 2 doses, nor evidence that a low nadir CD4+ T-cell count compromised responses. Post-third-dose humoral responses substantially exceeded post-second-dose levels, though Omicron-specific responses were consistently weaker than responses against wild-type virus. Nevertheless, post-third-dose responses in PLWH were comparable to or higher than controls. An mRNA-1273 third dose was the strongest consistent correlate of higher post-third-dose responses. CONCLUSION: PLWH receiving suppressive antiretroviral therapy mount strong antibody responses after 2- and 3-dose COVID-19 vaccination. Results underscore the immune benefits of third doses in light of Omicron.

Antibody response durability following three-dose coronavirus disease 2019 vaccination in people with HIV receiving suppressive antiretroviral therapy.

BACKGROUND: Limited data exist regarding longer term antibody responses following three-dose coronavirus disease 2019 (COVID-19) vaccination, and the impact of a first SARS-CoV-2 infection during this time, in people with HIV (PWH) receiving suppressive antiretroviral therapy (ART). We quantified wild-type-specific, Omicron BA.1-specific and Omicron BA.5-specific responses up to 6 months post-third dose in 64 PWH and 117 controls who remained COVID-19-naive or experienced their first SARS-CoV-2 infection during this time. DESIGN: Longitudinal observational cohort. METHODS: We quantified wild-type-specific and Omicron-specific anti-Spike receptor-binding domain IgG concentrations, ACE2 displacement activities and live virus neutralization at 1, 3 and 6 months post-third vaccine dose. RESULTS: Third doses boosted all antibody measures above two-dose levels, but BA.1-specific responses remained significantly lower than wild-type-specific ones, with BA.5-specific responses lower still. Serum IgG concentrations declined at similar rates in COVID-19-naive PWH and controls post-third dose (median wild-type-specific and BA.1-specific half-lives were between 66 and 74 days for both groups). Antibody function also declined significantly yet comparably between groups: 6 months post-third dose, BA.1-specific neutralization was undetectable in more than 80% of COVID-19 naive PWH and more than 90% of controls. Breakthrough SARS-CoV-2 infection boosted antibody concentrations and function significantly above vaccine-induced levels in both PWH and controls, though BA.5-specific neutralization remained significantly poorer than BA.1 even post-breakthrough. CONCLUSION: Following three-dose COVID-19 vaccination, antibody response durability in PWH receiving ART is comparable with controls. PWH also mounted strong responses to breakthrough infection. Due to temporal response declines, however, COVID-19-naive individuals, regardless of HIV status, would benefit from a fourth dose within 6 months of their third.

Clinical Outcomes and Quantitative HBV Surface Antigen Levels in Diverse Chronic Hepatitis B Patients in Canada: A Retrospective Real-World Study of CHB in Canada (REVEAL-CANADA).

BACKGROUND: Hepatitis B surface antigen (HBsAg) loss is associated with improved clinical outcomes for individuals with chronic hepatitis B (CHB); however, the effects of varying HBsAg levels on clinical outcomes in diverse cohorts are understudied. METHODS: In this cross-sectional, multicentre, retrospective study, the data on adult subjects enrolled in the Canadian HBV Network with CHB seen from 1 January 2012 to 30 January 2021 with the treatment and virologic data within 1 year of HBsAg testing were analyzed. Patients were tested for HBsAg using qualitative (for HBsAg-negative samples) and/or commercial quantitative assays. Fibrosis or hepatic necroinflammation was determined by the liver stiffness measurement (LSM). The baseline data were summarized using descriptive statistics and compared by using univariable/multivariable analyses. RESULTS: This study included 844 CHB patients, with a median age of 49.6 years (IQR 40.1-60.5), and 37% were female. In total, 751 patients (78.6%) had known ethnicity data, and 76.7% self-reported as Asian, 11.4% as Black, 6.8% as White, and 4.8% as other. Among the 844 patients, 237 (28.0%) were HBsAg (-) (1000 IU/mL. Overall, 80% (682) had known HBeAg status at the last follow-up, and the majority (87.0%) were HBeAg-negative. In addition, 54% (461/844) had prior antiviral therapy, 19.7% of which (16.3, 23.7, n = 91) were HBsAg (-). The treated patients had a lower risk of cirrhosis (16.46, 95% CI 1.89-143.39, p = 0.01) or HCC (8.23, 95% CI 1.01-67.39, p = 0.05) than the untreated patients. A lower proportion of the HBsAg-loss group had cirrhosis (5.7% vs. 10.9%, p = 0.021) and HCC (0.9% vs. 6.2%, p = 0.001). CONCLUSION: In this retrospective, ethnically diverse cohort study, CHB patients who received antiviral therapy and/or had HBsAg loss were less likely to develop cirrhosis and HCC, confirming the results of the studies in less diverse cohorts. No association was found between the qHBsAg level and fibrosis determined with LSM. Individuals who achieved HBsAg loss had low-level qHBsAg within 1 year of seroclearance.

Serial infection with SARS-CoV-2 Omicron BA.1 and BA.2 following three-dose COVID-19 vaccination.

SARS-CoV-2 Omicron infections are common among individuals who are vaccinated or have recovered from prior variant infection, but few reports have immunologically assessed serial Omicron infections. We characterized SARS-CoV-2 humoral responses in an individual who acquired laboratory-confirmed Omicron BA.1.15 ten weeks after a third dose of BNT162b2, and BA.2 thirteen weeks later. Responses were compared to 124 COVID-19-naive vaccinees. One month post-second and -third vaccine doses, the participant's wild-type and BA.1-specific IgG, ACE2-displacement and virus neutralization activities were average for a COVID-19-naive triple-vaccinated individual. BA.1 infection boosted the participant's responses to the cohort ≥95th percentile, but even this strong "hybrid" immunity failed to protect against BA.2. Reinfection increased BA.1 and BA.2-specific responses only modestly. Though vaccines clearly protect against severe disease, results highlight the continued importance of maintaining additional protective measures to counteract the immune-evasive Omicron variant, particularly as vaccine-induced immune responses naturally decline over time.

Automated Library Construction and Analysis for High-Throughput Nanopore Sequencing of SARS-CoV-2.

BACKGROUND: To support the implementation of high-throughput pipelines suitable for SARS-CoV-2 sequencing and analysis in a clinical laboratory, we developed an automated sample preparation and analysis workflow. METHODS: We used the established ARTIC protocol with approximately 400 bp amplicons sequenced on Oxford Nanopore's MinION. Sequences were analyzed using Nextclade, assigning both a clade and quality score to each sample. RESULTS: A total of 2179 samples on twenty-five 96-well plates were sequenced. Plates of purified RNA were processed within 12 h, sequencing required up to 24 h, and analysis of each pooled plate required 1 h. The use of samples with known threshold cycle (Ct) values enabled normalization, acted as a quality control check, and revealed a strong correlation between sample Ct values and successful analysis, with 85% of samples with Ct < 30 achieving a "good" Nextclade score. Less abundant samples responded to enrichment with the fraction of Ct > 30 samples achieving a "good" classification rising by 60% after addition of a post-ARTIC PCR normalization. Serial dilutions of 3 variant of concern samples, diluted from approximately Ct = 16 to approximately Ct = 50, demonstrated successful sequencing to Ct = 37. The sample set contained a median of 24 mutations per sample and a total of 1281 unique mutations with reduced sequence read coverage noted in some regions of some samples. A total of 10 separate strains were observed in the sample set, including 3 variants of concern prevalent in British Columbia in the spring of 2021. CONCLUSIONS: We demonstrated a robust automated sequencing pipeline that takes advantage of input Ct values to improve reliability.